An essential function of the vascular endothelium is to provide a semi- selective cellular barrier between the blood and the interstitial space of all organs. A variety of physical, inflammatory and bioactive stimuli alter the endothelial cell barrier thereby increasing vessel permeability and compromising organ function. In the setting of acute lung injury, the physiological consequence of endothelial cell gap formation and barrier dysfunction are alveolar flooding and hypoxemia resulting in marked increased in patient morbidity and mortality. In this program project, we will utilize well established in vitro models of endothelial cell permeability and examine multiple physiologic, biochemical and molecular determinants to better understand the regulation of the vascular barrier. Each project is based upon our working model that barrier function is governed by a dynamic equilibrium between competing contractile forces (driven by an acto-myosin) molecular motor) which promotes gap formation and barrier dysfunction and tethering forces which protect the integrity of the barrier. Project 1 will examine the molecular regulation of a novel myosin light chain kinase (MLCK) isoform which is key to contraction- mediated endothelial cell permeability. Project 2 will investigate important MLCK-independent mechanisms of barrier dysfunction including reductions in the activity of the barrier dysfunction which occurs in inflammatory lung settings as a consequence of neutrophil-endothelial cell interaction. Novel endothelial cell signaling pathways induced by neutrophil-derived extracellular phosphatidic acid and oxidants (including protein kinase C and tyrosine kinases) will be examined in relationship to the evoked endothelial cell force generation, monolayer resistance and barrier dysfunction. This multi-faceted program project approach will more quickly be able to answer fundamental questions regarding vascular permeability regulation. Important insights derived from these studies in clinically and physiologically relevant models may lead to novel strategies which limit vascular leak, preserve lung function and reduce morbidity/mortality from inflammatory lung injury.